A Culture-Free Platform for Rapid Pathogen Identification Project Summary Sepsis is a one of the leading causes of morbidity and mortality in the US, with more than 751,000 confirmed cases occurring every year and 210,000 mortalities. This high mortality rate (~30%) means that nearly 600 patients a day succumb to sepsis-related infections. In addition to the human toll, the economic burden is likewise immense, reaching $24B/year in the US, with an average cost of over $23,000 per patient. The key to treating sepsis properly is rapid and accurate identification of the causative microorganism (bacterial or fungal). Unfortunately, given the wide range of potential pathogens which can induce sepsis, the 'gold-standard' today for diagnosis remains culturing; which typically requires up to 3-5 days for accurate identification leading to a significant delay in the initiation of a targeted treatment protocol. The objective of this Small Business Innovation Research Phase I project is to demonstrate feasibility of a novel, culture- free assay for detecting and identifying the most clinically prevalent sepsis inducing fungal pathogens in a matter of hours. The proposed innovation in this project removes the need for time consuming and insensitive culturing, thus significantly decreasing the time from specimen to result from days to a few hours. Additionally as species-specific amplification is not required, our process is faster and is directly amicable to multiplexing. Our new process utilizes a new synthetic nucleic acid analogue, gamma-PNA, which has the demonstrated ability to invade dsDNA with unmatched sequence specificity through standard Watson-Crick base pairing rules. Our focus in this application is a specific proof-of-concept to be achievable within the scope of a Phase I project. The performance goal of this Phase I is to establish an assay employing gamma-PNA probes, capable of identifying and distinguishing among the most prominent sepsis inducing fungal pathogens. Furthermore, after successfully demonstrating our assay, we will titrate the assay down to 10 CFU. We will culminate our study by spiking the samples with increasing amounts of background human DNA, establishing that real-world levels of non-target DNA do not hinder the diagnostic process. Having achieved these goals, in Phase II we will develop a prototype system, working directly from phlebotomy samples and address all issues such as optimization, automation and commercial format. Given the ease in which our approach can be multiplexed, future efforts will be made to expand the repertoire of our assay by increasing the number of pathogens (to both bacterial and fungal) in our panel ultimately leading to the development of a 'one-assay system' for the culture-free identification of the ~20 most prevalent microbial pathogens which induce a septic response. Gamma-PNA probes are an attractive approach for pathogen identification as they offer remarkable sequence specificity as well as extremely high target affinity facilitating a more sensitive and more specific readout. The proposed assay would enable a dramatically faster, simplified and more accurate method of diagnosing fungal blood stream infections, enabling a low-cost method to rapidly and efficiently reduce the unbearably high mortality levels currently associated with sepsis.